Development of benzodiazepine and picrotoxin (t-butylbicyclophosphorothionate) binding sites in rat cerebellar granule cells in culture.
The specific bindings of [3H]flunitrazepam [( 3H]FLU), [3H]CGS 8216, and t-[35S]butylbicyclophosphorothionate [( 35S]TBPS) to sites on rat cerebellar granule cells all increase from 4 to 15 days in culture, although their time courses differ. Specific [3H]FLU binding doubles, [3H]CGS 8216 binding triples, and [35S]TBPS binding increases about fourfold from 4 to 15 days in culture. Displacement studies, using the type I-selective ligand CL 218,872, indicate that at 4 days the [3H]FLU binding sites are almost entirely "type II," judging from an IC50 value near 300 nM and a pseudo-Hill number near 1. By 10 days, approximately equal numbers of type I and type II binding sites are present in the cultured cells, and this ratio remains constant thereafter (12 and 15 days). At days 10-15, both the IC50 value for CL 218,872 (near 100 nM) and the pseudo-Hill number (near 0.7) remain constant and are significantly different from the values at culture day 4. The development of specific [35S]TBPS binding parallels that of [3H]CGS 8216 binding more closely than the development of [3H]FLU binding. The [3H]CGS 8216/[3H]FLU ratio increased by a factor of 1.6 from day 4 to day 15 (p less than 0.001). Taken together, our data suggest the existence of several gamma-aminobutyric acidA (GABAA) receptor subunits, the relative proportions of which change during development. The presence of the GABA-mimetic 4,5,6,7-tetrahydroisoxazolo[5,4c]pyridine-3-ol (THIP) in the culture medium had no apparent effect on any of the binding sites studied, although THIP was shown previously to induce low-affinity GABA binding sites.
Squires
R F
RF
Nathan Kline Institute for Psychiatric Research, Orangeburg, NY 10962.
Saederup
E
E
Damgaard
I
I
Schousboe
A
A
D000818:Animals; D001569:Benzodiazepines / Q000037:antagonists & inhibitors; D001665:Binding Sites; D001643:Bridged Bicyclo Compounds / Q000378:metabolism*; D019086:Bridged Bicyclo Compounds, Heterocyclic; D001952:Bridged-Ring Compounds / Q000378:metabolism*; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology / Q000378:metabolism*; D005445:Flunitrazepam / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006098:Granulocytes / Q000378:metabolism*; D007555:Isoxazoles / Q000494:pharmacology; D011720:Pyrazoles / Q000378:metabolism*; D011724:Pyridazines / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology; D051381:Rats
D001643:Bridged Bicyclo Compounds; D019086:Bridged Bicyclo Compounds, Heterocyclic; D001952:Bridged-Ring Compounds; D007555:Isoxazoles; D011720:Pyrazoles; D011724:Pyridazines; D011725:Pyridines; D001569:Benzodiazepines; D005445:Flunitrazepam; C021604:CL 218872; C037476:tert-butylbicyclophosphorothionate; C055334:iso THIP; C033830:2-phenylpyrazolo(4,3-c)quinolin-3(5H)-one